Nicotinamide adenine dinucleotide (NAD) is a critical cofactor for numerous oxidoreductase reactions that interconvert the oxidized form (NAD+) and the reduced form (NADH). Several enzymatic reactions can also consume NAD, breaking the glycolytic bond between the nicotinamide (NAM) and ADP-ribose moieties. For example, the Sir2 family of NAD-dependent protein deacetylases (the Sirtuins) couple the deacetylation of specific acetyl-lysine side chains with NAD hydrolysis. Importantly, the liberated NAM can cause strong feedback inhibition of deacetylation. The Saccharomyces cerevisiae Sir2 protein is a histone deacetylase that is required for gene silencing and for maintaining a long replicative lifespan. Much of the research effort on mammalian Sirtuins has focused on the SIRT1 protein, which is the human Sirtuin most similar to yeast Sir2. SIRT1 deacetylates transcription factors such as p53, FOX03, or NF-KappaB, which helps decide cell survival in response to a particular cellular stress or extracellular signal, via the inhibition or potentiation of apoptosis. Genetic studies have determined that manipulations of a nuclear NAD salvage pathway in yeast can modulate Sir2-mediated silencing and longevity activity by maintaining high intracellular NAD concentration and by limiting the NAM concentration. Together these findings raise the intriguing possibility that Sir2 is a direct link between the cell metabolic status (via the NAD concentration, NAD/NADH ratio, or NAM concentration) and specific protein deacetylation. Therefore, the long-term goals of this research project are to determine how NAD salvage/synthesis in the nucleus changes in response to cellular growth conditions and how this translates into the regulation of Sir2 activity in yeast and SIRT1 activity in human cells. Analysis of the human pathways will be guided by information learned from the yeast system. The first specific aim will employ genetic methods to further dissect how yeast cells respond, to and compensate for, high intracellular and extracellular NAM concentrations. Experiments in the second aim are tightly focused on directly testing the hypothesis that NAD synthesis/salvage and NAM clearance in the nucleus is critical for yeast Sir2-mediated silencing and longevity. In the third aim, the specific DNA synthesis or salvage pathways responsible for regulating human SIRT1 will be identified and characterized in the context of the known SIRT1 deacetylase targets, p53 and NF-KappaB. Together these yeast-inspired experiments will help identify new potential targets for cancer therapeutics or anti-aging interventions.